Systematic investigation of recipient cell genetic requirements reveals important surface receptors for conjugative transfer of IncI2 plasmids

Bacterial conjugation is a major horizontal gene transfer mechanism. While the functions encoded by many conjugative plasmids have been intensively studied, the contribution of recipient chromosome-encoded genes remains largely unknown. Here, we analyzed the genetic requirement of recipient cells for conjugation of IncI2 plasmid TP114, which was recently shown to transfer at high rates in the gut microbiota. We performed transfer assays with ~4,000 single-gene deletion mutants of Escherichia coli. When conjugation occurs on a solid medium, we observed that recipient genes impairing transfer rates were not associated with a specific cellular function. Conversely, transfer assays performed in broth were largely dependent on the lipopolysaccharide biosynthesis pathway. We further identified specific structures in lipopolysaccharides used as recipient cell surface receptors by PilV adhesins associated with the type IVb accessory pilus of TP114. Our strategy is applicable to study other mobile genetic elements and understand important host cell factors for their dissemination.


LPS structure description
The outer membrane of Gram-negative bacteria is an essential feature acting like a barrier to protect cells from toxic compounds such as antibiotics and detergents 1 .
From the outer membrane to the extracellular space, the LPS structure consists of (i) lipid A, a hydrophobic glycolipid which anchors LPS in the outer membrane; (ii) core oligosaccharide (core-OS), a non-repeating oligosaccharide containing sugars such as heptose and keto-deoxyoctulosonate (Kdo); and (iii) O antigen, a polysaccharide made of up to 50 repeating oligosaccharide subunits (Fig. 3 c).
LPS biosynthesis involves a large number of enzyme activities, governed by more than 40 genes 2,3 that account for a large structural diversity.In the Enterobacteriaceae, the core-OS is divided into two distinct regions, the inner and the outer core-OS (Fig. 3 c).The inner core is highly conserved and comprises three Kdo and L-glycero-D-manno-heptose (Hep) residues and is often phosphorylated.The outer core-OS comprises a tri-hexose backbone that can be modified with varying side-branch substitutions 4 .Escherichia coli serotypes are determined by the type of polysaccharide antigens on their cell membrane including the O antigen.A total of ~178 distinct antigens have been formally defined for E. coli alone.This diversity allows bacteria to have a surface that offers a selective advantage in its specific niche 3 .Despite this high degree of diversity in O antigen, only five distinct core-OS structures are found in E. coli, designated K-12, R1, R2, R3 and R4 (Fig. 5 b).These core-OS structures are synthesized through the successive addition of various sugars to lipid A by the products of the waa genes (formerly rfa genes) 4 .

PilV variants recognize receptor structures in the core-OS of E. coli LPS
The loss of long O16 antigen in E. coli BW25113 (K-12 core-oligosaccharide) is due to the disruption of the rhamnosyl transferase wbbL gene with an IS5 element, termed the rfb-50 mutation 1,5 .Since the complementation of this lesion by insertion of an intact wbbL gene from an O antigen-expressing strain of E. coli into the chromosome of E. coli BW25113 restored O antigen expression 1,6 , all other genes are functional.To ensure that these other genes do not incorporate new sugars into the LPS structure, we generated an E. coli BW25113 mutant in which the rfbABCD, wzxB, glf and wbbHIJKL genes were removed (Fig. 3 c).This ΔO antigen mutant, exhibiting a core-OS, as well as a mutant displaying only the lipid IVA, called ClearColi 7 (Fig. 3 c), were subjected to pairwise conjugation in broth using all TP114 derivatives bearing a single PilV variant 8 (Fig. 3 d).As previously reported 8 , transfer rates of TP114 mutant in which the variable region of pilV was replaced by a FLAG tag was virtually unaffected on solid medium, but its capacity to conjugate in broth was severely altered.Moreover, since E. coli BW25113 and BW25113ΔO antigen were recognized by the same three adhesins (PilVA', PilVC and PilVC') while ClearColi did not generate any transconjugants, we can infer that these adhesins -and potentially those that did not recognize the K-12 core structure-exhibit specificities for a receptor present in the core-OS.

Elucidation of receptor structure(s) for PilV variants using knockout mutants
Results surrounding the elucidation of receptor structures of PilVA' and PilVC are described in detail in the main article and will not be elaborated here.The use of a donor strain expressing only one PilV variant at a time 8 allowed us to study their specificities towards different recipient bacteria.The pairwise mating experiments in broth allowed us to validate receptor structures since strains displaying the receptor structure at the surface of the cell generated transconjugants, while those missing the receptor molecule do not.For example, the PilVA adhesin of plasmid R64 was shown to specifically bind to N-acetylglucosamine-β-(1-3)-glucose 9 (Fig. 5 b).This receptor structure is only present in E. coli R1 and thus this strain is the only one producing transconjugants when mating with TP114-bearing PilVA adhesin, which is homologous to R64 PilVA.Moreover, it was shown that a ΔwaaL knockout mutant of E. coli R1 was no longer recognized by PilVA adhesin 9 .
Consequently, we expect that cloning the genes responsible for the addition of Nacetylglucosamine-β-(1-3)-glucose disaccharide and their expression in a strain representing one of the other core-OS would produce transconjugants when mating with a donor expressing PilVA adhesin.
The conjugation results with the PilVC' variant were also in agreement with the receptor structure proposed for the homolog of R64 as being glucose-α-(1-2)glucose or glucose-α-(1-2)-galactose. Indeed, in the case of E. coli R2 and E. coli BW25113 which displays the K-12 core-OS, the glucose-α-(1-2)-glucose is the target of PilVC' adhesin.Effectively, E. coli BW25113ΔwaaR, missing the third glucose, is no longer recognized by PilVC' adhesin (Supp.Fig. 4 b).Moreover, all other knockout mutants affecting the core-OS lower in the structure, such as ΔwaaO (Supp.Fig. 4 c), ΔwaaG, ΔwaaF, ΔwaaC (Fig. 4 f, g, h) does not generate transconjugants when mating in broth with a donor harbouring the PilVC' adhesin (Fig. 5 a).On the other hand, in E. coli R1 and R4, it is the glucose-α-(1-2)galactose that acts as a receptor structure (Fig. 5).One intriguing thing about those receptors is that both can be found in the outer core-OS of E. coli R3, but this strain does not produce any transconjugants when using the PilVC' adhesin.This could mean that the glucose added by WaaG may be important in the receptor structure.
To investigate this possibility, the waaO and waaR genes could be introduced in trans in E. coli BW25113 ΔwaaG.If this strain, harbouring a chimeric LPS, is still recognized by the PilVC' adhesin, then the receptor is a disaccharide, but if not, the receptor could be a trisaccharide.

Elucidation of receptor structure(s) for PilV variants using knock-in mutants
To explore other potential receptor structures, we cloned one or two waa genes originating from another core-OS prototype in an arabinose inducible plasmid (pBAD30).For example, when introducing pWaaOX (bearing waaO and waaX genes from E. coli R4) in E. coli R3, this new strain expressing chimeric LPS produced transconjugants when mating in broth with the PilVC' adhesin, which was not the case with the wild type strain (Supp.Fig. 4 d, e).Since waaO and waaX genes are responsible for the incorporation of the galactose-β-(1-4)-glucose in the LPS, this disaccharide is likely a new receptor structure of PilVC' adhesin.
In our results, only the R2 core LPS and strains in which we added the appropriate genes were recognized by the PilVB variant.More precisely, when introducing waaK in trans in E. coli BW25113 or waaK along with waaR genes in E. coli R4 (Supp.Fig. 4 f, g, h), those strains produced transconjugants when mating in broth with a donor strain harbouring PilVB adhesin, while the wild type strains did not (Supp.Fig. 7).Since waaK is responsible for the addition of an Nacetylglucosamine sugar on a glucose molecule with an α-(1-2) linker, this modification could occur on the first glucose of BW25113 LPS, thus allowing transconjugants to form upon mating assays in broth using the PilVB adhesin (Fig. 4).This again highlights the fact that the nature of the linker between the two molecules is crucial since there is already an N-acetylglucosamine-α-(1-6)-glucose in the O16 antigen of E. coli BW25113 5 , but the PilVB adhesin does not recognize the wild-type strain.N-acetylglucosamine-α-(1-2)-glucose structure was previously proposed as the target of PilVB' for R64 10 .However, our results show that Nacetylglucosamine-α-(1-2)-glucose is rather the receptor structure for the PilVB variant.
The introduction of pWaaD, pWaaT, pWaaV, pWaaW, pWaaJ, pWaaVL, pWaaRK, pWaaTW-R1 or pWaaTW-R4 into E. coli BW25113 and expression of chimeric LPS did not change the pattern of PilV adhesin recognition of the strains (Supp.Fig. 7).Underline represent binding nucleotides plates using the Singer Rotor HDA (e).f, After 6h of conjugation at 37°C, mating plates were replicated using the Singer Rotor HDA on two selection plates, containing kanamycin and chloramphenicol to select the transconjugants (deletion mutants that have acquired TP114) or kanamycin only to select recipient bacteria.

Supplementary
To do so, each conjugation was replicated 16 times sequentially to create a serial dilution array to be able to assess the transfer rate for each mutant.Plates are then incubated at 37°C overnight before being imaged (g), and the growth of each mutant is converted into a density value.Finally, a ratio between transconjugant density and recipient density is made to determine the conjugation score of each mutant.
with the E. coli BW25113∆yejO recipient strain to vary donor to recipient ratios.After a 6h conjugation time, transconjugants (Kn and Cm) and recipients (Kn) were selected and the transfer rate was measured as described in the manuscript.(f) TP114 variants with different transfer rates were used in the standard manual conjugation assay or high-throughput screening approach.The TP114 variants were hosted in E. coli MG1655Nx R as the donor and were transferred to the E. coli BW25113∆yejO recipient strain in broth using the high-throughput conjugation assay.The results suggest that conjugation scores track relatively well with manual transfer rates with a detection limit around 10 -5 .
the conjugation results underneath 5.9 x 10 -5 were not significant and were identified with a cross mark as well.

Table 1 . Strains and plasmids used in this study.
R BW25113 where O antigen (rfbABCD, wzxB, glf, wbbHIJKL) have been replace by Kn R by recombineering Supplementary